CD36/Src/ERK通路参与单核细胞向巨噬细胞的分化

CD36/Src/ERK pathway is involved in process of monocyte differentia⁃tion into macrophages

目的:探讨脂肪酸转位酶(fatty acid translocase,FAT/CD36)在单核细胞向巨噬细胞分化中的作用。方法:采用不同浓度(0、100和200μg/L)佛波酯(phorbol 12-myristate 13-acetate,PMA)诱导人源单核细胞THP-1向巨噬细胞分化;用CD36小片段干扰RNA(CD36 small interfering RNA,siCD36)干扰THP-1细胞CD36的表达;又用CD36 cDNA的重组慢病毒转染THP-1细胞,构建CD36过表达细胞系。显微镜下观察细胞形态;结晶紫染色法检测细胞黏附能力;流式细胞术和Western blot检测细胞分化过程中CD36的蛋白表达,real-time PCR检测CD36、CD11b和CD80的mRNA水平。Western blot检测THP-1细胞向巨噬细胞分化过程中细胞外信号调节激酶(extracellular signal-regulated kinase,ERK)和Src酪氨酸激酶的表达。结果:THP-1细胞向巨噬细胞分化的过程中,CD36表达增加(P<0. 01)。与正常细胞相比,siCD36干扰细胞的表面积减小,黏附能力降低(P<0. 01),细胞中CD11b和CD80的mRNA水平下降(P<0. 01);而CD36过表达细胞的表面积明显增大,黏附能力增强(P<0. 05),且细胞中CD11b和CD80的mRNA水平升高(P<0. 01)。THP-1细胞向巨噬细胞分化过程中,ERK的磷酸化明显增加,而siCD36干扰的THP-1细胞分化过程中ERK的磷酸化和Src的磷酸化则明显减少(P<0. 05)。结论:CD36通过增强Src磷酸化从而促进ERK的磷酸化及活化,最终促进THP-1细胞向巨噬细胞分化。

AIM:To investigate the role of fatty acid translocase(FAT/CD36)on differentiation of monocytes to macrophages.METHODS:Human monocyte THP-1 cells were treated with phorbol 12-myristate 13-acetate(PMA)at 0,100 and 200μg/L.Small interfering RNA(siRNA)targeting CD36(siCD36)was employed to knock down the expres⁃sion of CD36 in THP-1 cells.The CD36 over-expression(CD36OE)cell line was constructed by transfection with a recom⁃binant lentivirus containing CD36 cDNA.Optical microscopy and crystal violet staining were used to detect the monocyte morphological changes and adhesion ability.The protein expression of CD36 was measured by flow cytometry and Western blot.The mRNA levels of CD36,CD11b and CD80 were detected by real-time PCR.The protein levels of extracellular signal-regulated kinase(ERK)and Src tyrosine kinase were determined by Western blot.RESULTS:The cellular adhe⁃siveness of THP-1 cells was elevated in the process of monocytes differentiation,and the expression of CD36 was increased in this process as well(P<0.01).siCD36 was transfected into the THP-1 cells(CD36i group)and the silencing efficiency was approximately 80%.The cell surface area and cellular adhesiveness were significantly decreased in CD36i group com⁃pared with scrambled siRNA(NCi)group(P<0.01).The mRNA levels of CD11b and CD80 were decreased in CD36i group compared with NCi group(P<0.01).The cell surface area and cellular adhesiveness were increased in CD36OE group compared with empty vector(vector)group(P<0.05).The mRNA levels of CD11b and CD80 were increased in CD36OE group compared with vector group(P<0.01).The phosphorylation levels of ERK and Src were decreased in CD36i group compared with NCi group(P<0.05).CONCLUSION:CD36 promotes the differentiation of human mono⁃cyte THP-1 cells to macrophages by increasing the phosphorylation of Src and further activating ERK.